Ion Hydration Dynamics in Conjunction with a Hydrophobic Gating Mechanism Regulates Ion Permeation in p7 Viroporin from Hepatitis C Virus

The selectivity of the p7 channel from hepatitis C virus (HCV) toward K⁺ over Ca²⁺ has made the channel an intriguing system for investigating ion permeation. The present study employs umbrella sampling free energy calculations to investigate the atomistic details of cation conduction through the channel. The free energy profiles suggest that the energy barrier for Ca²⁺ conduction is higher than that for K⁺ conduction by about 4.5 kcal/mol, thus explaining the selectivity exhibited by the channel toward K⁺. A hydrophobic stretch in the channel is proposed to be the primary factor that discriminates K⁺ from Ca²⁺, and the ion solvation dynamics in this stretch reveals interesting insights into the atomistic mechanisms involved. Two-dimensional free energy landscapes for the ion permeation reveal differences in the lateral motions of K⁺ and Ca²⁺ with respect to the pore axis, and provide additional details of ion–protein interactions that govern selectivity

Urea–Aromatic Stacking and Concerted Urea Transport: Conserved Mechanisms in Urea Transporters Revealed by Molecular Dynamics

Urea transporters are membrane proteins that selectively allow urea molecules to pass through. It is not clear how these transporters allow rapid conduction of urea, a polar molecule, in spite of the presence of a hydrophobic constriction lined by aromatic rings. The current study elucidates the mechanism that is responsible for this rapid conduction by performing free energy calculations on the transporter dvUT with a cumulative sampling time of about 1.3 μs. A parallel arrangement of aromatic rings in the pore enables stacking of urea with these rings, which, in turn, lowers the energy barrier for urea transport. Such interaction of the rings with urea is proposed to be a conserved mechanism across all urea-conducting proteins. The free energy landscape for the permeation of multiple urea molecules reveals an interplay between interurea interaction and the solvation state of the urea molecules. This is for the first time that multiple molecule permeation through any small molecule transporter has been modeled

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

<div><p>The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.</p> </div

Representative structures.

<p>The structures for the different oligomeric states before (top row) and after (bottom row) 10 ns simulation in an implicit membrane environment are shown.</p

Interhelical distance and protein-lipid interactions.

<p>(A) Probability distribution of interhelical distance for tetramer, pentamer and hexamer. The distance between the centres-of-mass of adjoining helices was calculated. Only the helical backbone was considered, and the top three and bottom three residues were neglected. (B) Average number of hydrogen bonds between lipid headgroups and polar residues for Arg30 and headgroup (left panel), and Tyr29 and headgroup (right panel). The cutoffs used were 3.5 Å for the donor-acceptor distance, and 45° for the donor-hydrogen-acceptor angle.</p

Pore profile.

<p>(A) View along the pore axis from the C-terminal showing the Ser23 residue in “licorice” representation. Serine faces the interior of the channel in the pentamer model. (B) Side view of the pentamer model showing the location of the Ser23 residue (in “licorice” representation) and water molecules in the pore. The N-terminal side is on the top and the C-terminal is at the bottom. (C) Pore radius across the axis of the pentamer model. The pore is constricted towards the N-terminal side (top half).</p

Atomistic Detailed Mechanism and Weak Cation-Conducting Activity of HIV-1 Vpu Revealed by Free Energy Calculations

<div><p>The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na⁺ and K⁺ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.</p></div

Structural features of the pentamer model.

<p>(A) Kink around the Ile17 residue in the pentamer model. (B) The three residues known to interact with tetherin shown in van der Waals representation.</p

Replica-exchange molecular dynamics in an implicit membrane environment.

<p>(A) Probability distribution of the tilt angle for the conformations sampled at 300 K from the last 9 ns of replica-exchange molecular dynamics. (B) Average potential energy and (C) free energy of the different oligomeric states over the last 9 ns of replica-exchange molecular dynamics. The values shown are relative to the monomer. (D) RMSD of the tetramer, the pentamer, and the hexamer in the REX/MD simulations.</p

Molecular dynamics in an explicit membrane environment.

<p>(A) Models for the tetramer, pentamer and hexamer after simulation in a fully hydrated lipid bilayer. The images for the pentamer are after 30 ns, and those for the tetramer and hexamer are after 10 ns. The lipid bilayer and solvent molecules have been omitted for clarity. The pentamer retained a channel-like structure in both the simulations. (B) RMSD of the different oligomeric states.</p